Driver mutations in Isocitrate Dehydrogenase 1 (IDH1) are present in 70-80% of grade II and III gliomas, with the majority eventually progressing to glioblastoma multiforme (GBM). In this molecularly distinct class of malignant gliomas, mutant IDH1 enzyme produces 2-hydroxyglutarate (2-HG), an oncometabolite that inhibits ?-ketoglutarate dependent histone and DNA demethylases resulting in characteristic hypermethylation of genomic DNA and suppression of cellular differentiation. We have demonstrated the preclinical efficacy and mechanism of action of the approved DNA demethylating drug 5-Azacytidine (5-Aza) and revealed combinations that further enhance survival. In models with the native IDH1 mutation, 5-Aza administration reduces tumor burden, extends survival and induce differentiation in vivo. Combination of 5-Aza with standard of care, use of differentiation, and pro-apoptotic drugs all show further benefit. The focus of this revised grant is to optimize treatment and validate mechanism tailored to either grade II or high grade IDH1 mutant gliomas. In Aim 1 we propose to demonstrate the mechanism of 5-Aza induced cell growth reduction and further optimize 5-Aza with standard of care treatment for IDH1 mutant glioma. We have recently demonstrated reduction in tumor burden and extend survival in our IDH1 mutant PDX model works better when using 5-aza in combination with temozolomide regardless of the order of administration. In Aim 2 we will determine the impact of DNA demethylating agent in combination with differentiation therapy using retinoic acid in IDH1 mutant glioma. Our preliminary data shows that 5-Aza regulates retinoic acid signaling, and in IDH1 mutant PDX glioma model the two work synergistically to slow tumor growth. In aim 3 we propose to combine 5-Aza with the proapoptotic drug PAC-1 in IDH1 mutant glioma and explore the mechanism of response. Preliminary data indicate that Procaspase Activating Compound-1 (PAC-1), a novel brain penetrant procaspase 3 cleaving small molecule, functions to increase apoptosis and survival in an intracranial model of IDH1 mutant glioma. Our unifying hypothesis for each of these aims is that the combination of the mechanisms of reversing pathological demethylation will enhance known anticancer mechanisms of DNA damage, cellular senescence and/or apoptosis, and that interdependent mechanisms will combine to safely increase survival. Each of the small molecule therapies proposed show evidence of single agent efficacy in preclinical studies for glioma. We propose to determine how the mechanisms of demethylation, differentiation therapy and caspase 3 activation best combine safely for a therapeutic response. Our major goal is to produce the preclinical data that might support a new trial for patients with IDH1 mutant glioma. Those therapies with the possibility of causing DNA damage will be reserved for high grade gliomas, while less toxic therapies are proposed for low grade gliomas, where there is the possibility of a large impact on survival benefit.